Metabolic plasticity, essentiality and therapeutic potential of ribose-5-phosphate synthesis in Toxoplasma gondii

Ribose-5-phosphate (R5P) is a precursor for nucleic acid biogenesis; however, the importance and homeostasis of R5P in the intracellular parasite Toxoplasma gondii remain enigmatic. Here, we show that the cytoplasmic sedoheptulose-1,7-bisphosphatase (SBPase) is dispensable. Still, its co-deletion with transaldolase (TAL) impairs the double mutant’s growth and increases 13C-glucose-derived flux into pentose sugars via the transketolase (TKT) enzyme. Deletion of the latter protein affects the parasite’s fitness but is not lethal and is correlated with an increased carbon flux via the oxidative pentose phosphate pathway. Further, loss of TKT leads to a decline in 13C incorporation into glycolysis and the TCA cycle, resulting in a decrease in ATP levels and the inability of phosphoribosyl-pyrophosphate synthetase (PRPS) to convert R5P into 5′-phosphoribosyl-pyrophosphate and thereby contribute to the production of AMP and IMP. Likewise, PRPS is essential for the lytic cycle. Not least, we show that RuPE-mediated metabolic compensation is imperative for the survival of the ΔsbpaseΔtal strain. In conclusion, we demonstrate that multiple routes can flexibly supply R5P to enable parasite growth and identify catalysis by TKT and PRPS as critical enzymatic steps. Our work provides novel biological and therapeutic insights into the network design principles of intracellular parasitism in a clinically-relevant pathogen.


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April 2023

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All studies must disclose on these points even when the disclosure is negative.For virulence test, blood samples collected from surviving animals were tested 30 days post-infection, and mice seronegative by immunofluorescence were excluded from further analysis.
All experiments were repeated at least with 2-3 biological replicates.All results were successfully replicated.
In vivo experiment, mouse numbers (n = 3 -10 in each group) were based on our previously published work and elsewhere in the literature.
Mice were randomly assigned to experimental groups.Randomization does not apply to our other experiments as we have been analyzing wild type parasites vs knock-out mutants, or experiments conducted by treating samples and controls side by side under the indicated conditions The plaque assays were performed in a blinded manner and confirmed independently by two different people.For other experiments, blinding was not needed because the data produced had clear endpoints that were not subjected to investigator bias.
data have been deposited and released with the accession number PRJNA947326 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA947326/).Transcriptomics sequencing data are available in the short read archive (SRA) of the database of the National Center for Biotechnology Information under the accession number PRJNA947324(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA947324/).The raw proteomics data have been deposited in the ProteomeXchange Consortium (www.proteomexchange.org)via the iProX partner repository with the accession number: PDX041014.All other data are presented in the article or the supplementary information.Source data are provided with this paper.Reference genome of the Toxoplasma GT1 strain: https://toxodb.org/toxo/app/record/dataset/NCBITAXON_507601.Since the study involves comparison between wild type parasites and knockout mutant parasites, a minimum of three biological replicates were chosen for each separate experiment.Sample sizes in other experiments were chosen to reflect biological and technical variance of the investigated parameters based based on previously published literature (1). 1. Jin-Lei Wang, Ting-Ting Li, Hany M. Elsheikha, Qin-Li Liang,Zhi-Wei Zhang, MengWang, L. David Sibley, Xing-Quan Zhu.The protein phosphatase 2A holoenzyme is a key regulator of starch metabolism and bradyzoite differentiation in Toxoplasma gondii.Nature Communications.2022 Dec 8;13(1):7560.